Protein Conformational Disorder
It has been recognized in the recent years that there is a class of diseases and disorders that correlates with the presence of aggregates, whether intra- or extra-cellular, of misfolded or conformationally altered proteins. These proteins exist in a non-diseased environment. In a disease state, however, through certain alterations in the conformation, they adopt a secondary/tertiary structure different from those in the non-diseased state. The amino acid sequence is often unaltered. The misfolded proteins tend to self-associate, aggregating in an ordered fashion, form toxic precipitates, and deposit into tissues. The aggregated protein often takes a fibrillar appearance.
Examples of these disorders, now known as “protein conformational disorders” (PCDs), include but are not limited to Alzheimer's disease (AD), Parkinson disease (PD), Type-2 diabetes, amyotrophic lateral sclerosis (ALS), dialysis-related amyloidosis (DRA), reactive amylosis, cystic fibrosis (CF), sickle cell anemia, Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD) and related disorders, and systemic and cerebral hereditary amyloidosis. Examples of globular proteins that undergo fibrillogenesis include transthyretin, beta 2 microglobulin, serum amyloid A protein, Ig light chains, insulin, human lysozyme, alpha lactalbumin, and monellin. Examples of natively unfolded proteins that undergo fibrillogenesis include amyloid beta protein, tau protein, alpha-synuclein, amylin, and prothymosin alpha.
Pathogenesis and Biochemical Progression of PCD
Investigators have correlated protein aggregate deposition with the degeneration of tissue. Although there remains controversy with regard to the “cause or effect” of the presence of aggregate and the manifestation of the disease pathology, evidence is accumulating that the pathology is caused by aggregates, perhaps by direct toxicity due to the aggregation or by a loss of biological function of the misfolded protein.
The formation of aggregates is referred to as “fibrillogenesis.” Before the start of fibrillogenesis, the protein relevant to PCD pathology is in a naturally folded conformation and in monomeric or defined oligomeric forms, each peptide comprising a mixture of alpha-helices, some beta-sheets, and random coils. By the end of fibrillogenesis, the protein is aggregated, and the peptide has adopted an altered conformation, i.e. mostly a beta-pleated sheet conformation. The conformational changes of the peptides and aggregation appear to coincide, but the cause and effect of conformational change and aggregation, and the sequence of events, remain to be elucidated.
When considering the pathogenesis of a PCD, it has been proposed that the fibrillogenesis is a crystallization-like process: after a “seed” of oligomers forms, an aggregate grows over time through self-association. The protein may take an altered conformation because the aggregate exists and serves as a template, or it may take the altered conformation because of other factors, but once in that conformation, easily participates in fibrillogenesis. In contrast, another proposal hypothesizes that the conformational alterations alone may not cause or promote aggregation, and there is a factor that induces the aggregation. Such underlying factors that promote or induce structural changes in the protein include inflammatory or oxidative environments, nitration, phosphorylation, pH, or metal ion exposure (high concentrations of copper ions can induce the oligomerization of β2 microglobulin monomers, which in turn leads to fibril formation (Eakin et al., Biochemistry 2004, 43, 7808-7815)).
Various treatment modes and possible therapeutic agents for PCDs are currently being investigated. Whether conformational change precedes the start of the fibrillogenesis or vice versa will influence the effectiveness of a treatment strategy. For example, a treatment mode with an assumption that fibrillogenesis is caused by the beta-sheet conformation will attempt to inhibit the beta-sheet formation. In contrast, if the assumption was that aggregation promotes further formation of proteins with a degenerative conformation, a treatment mode may aim to inhibit aggregation by various means. An illustration of the former approach includes an attempt to inhibit the formation of, or to break, beta-sheets, using peptides. Such peptides are designed from the sequences of areas of proteins most likely involved in the process of nucleation and aggregation, such as the hydrophic core of amyloid-beta, a peptide intimately involved in the pathology of Alzheimer's disease. An illustration of the latter approach is an attempt to manipulate protein conformation and prohibit nucleation and subsequent formation of amyloids, or, “amyloidogenesis,” by creating mini-chaperone peptides from outside of the beta-sheet regions.
Another promising approach, regardless of the mechanism of aggregate formation, is to focus on the aggregates themselves. There have been attempts to reduce the level of aggregated protein of interest by antibodies: given sufficient specificity and ability to promote clearance, an antibody has a potential to be an effective therapeutic. To overcome delivery challenges, attempts have been made to express such antibodies intracellularly from a delivered gene. However, despite its potential, currently, the existing antibody therapeutics, if any, do not sufficiently prevent, improve, or even slow progression of the pathology, and there remains largely unmet needs for an effective treatment for a PCD.
Strategy for Creating Synthetic Therapeutic Peptides
The development and exploitation of combinatorial chemistry (CC) has propelled drug discovery. Drug discovery can be generalized into two major steps, lead generation and lead optimization. Oftentimes, a lead compound is identified that has some of the desired characteristics of a commercially viable therapeutic, but has shortcomings such as a low specific activity, toxicity, instability, etc. Thus, once a lead is identified, practitioners attempt to optimize the lead compound by testing other related compounds with similar structures. CC allows practitioners to create and quickly screen a library made of a vast number of candidates, to identify those with a specific activity against a target of interest.
For peptide based drugs, the goal is to define a single, or a limited set of peptides which demonstrate a particular activity. The art of CC as applied to the synthesis of peptide libraries, too, has advanced, producing highly reliable and pure mixtures of peptides of great diversity. The process of identifying the single or limited set of peptides that were responsible for the observed activity from such diverse libraries, called deconvolution, is schematically represented in FIG. 1A.
An analogous process applies for development of a therapeutically effective antibody. Traditionally, antibodies were raised by immunizing an animal using a target protein or peptide as an antigen, either directly collecting sera for polyclonal antibodies (i.e. a mixture of antibodies enriched for those that bind to the target) or by creating hybridomas and selecting those hybridomas that produce monoclonal antibodies that bind to the target. In more recent years, phage display libraries have been used to present a large number of antibodies, from which antibodies that bind to the target is selected. In other words, antibody isolation is an initial screening of a lead molecule from a large number of candidates.
It is well known in the art, however, that an antibody that binds to the target is not necessarily one that has a desired therapeutic effect. As such, therapeutically effective antibodies may still have to be created through the process of lead optimization. The optimization may take a form of further screening of an antibody library (e.g. a phage display library), direct manipulation of complementarity determining regions of an immunoglobulin, or renewed immunization of an animal using related but different epitopes in an attempt to create a further variety in the enriched antibodies that the animal produces.
Low Immunogenicity, or Necessity for Highly Specific Antibodies
In PCD, for therapeutic, prophylactic, and diagnostic purposes, the antibodies that are desirable recognize and specifically bind to proteins of certain altered conformation. The difficulty lies in the fact that these proteins exist as normal parts of the patient's system, were it not for the altered conformation that they are in. Thus, even though these proteins are pathological, they may not elicit strong natural immune responses in the afflicted individuals, and it may be difficult to elicit an immune response (thus to raise antibodies) using the native sequence of the target protein in other subjects of the same species, or in an individual with similar immunological profile, which is often desirable due to the lower probability of adverse immunological reaction.
Another challenge is that the antibody should differentiate between the same protein in a non-pathological conformation and in a pathological conformation. A protein relevant to a PCD may have the same primary structure, whether in a non-pathological condition or in pathological condition. Without the ability to distinguish, the antibody intended for therapeutic purposes may adversely affect the patient by eliminating or interfering with the normal, functioning protein. Thus, a high specificity towards the particular conformation, or series of alterations, is required.
Although immunization with an immunogen having a single epitope may induce multiple antibodies having complementarity determining regions (CDR) different from each other, it may be difficult to strongly elicit (and thus detect and identify) all varieties of antibodies. In addition, even if antibodies are induced, the most easily inducible and detectable antibodies against such epitope may not include those antibodies with a high specificity towards the particular pathological conformation as described in the preceding paragraphs. In an attempt to overcome these challenges, investigators have designed peptides with sequences similar to the target peptides. These variations of the target peptides may induce generation of antibodies that are different from those induced by the target peptides, but may cross-react sufficiently with the target peptides. Thus, these related peptides may be desirable and/or required to identify an antibody that may not be induced by an epitope of the original sequence.
One such approach is the creation of altered peptide ligands (APL). This approach is schematically represented in FIG. 1B. An APL is defined as an analog peptide which contains a small number of amino acid changes from a starting sequence such as that of a native immunogenic peptide ligand. An illustrative example is an APL based on an epitope of myelin basic protein, MBP83-99 (ENPVVHEFKNIVTPRTP) (SEQ ID NO: 1), which is reported to be a target of autoimmune response causing multiple sclerosis. APLs were made by replacing the bold and underlined amino acid residues “E”, “N”, “E” and “K,” with various other amino acid residues. Screening for peptides that appeared to have the desired activity of neutralizing antibodies against MBP83-99 yielded a single peptide having the amino acid residue sequence AKPVVHLFANIVTPRTP (SEQ ID NO: 2), Kim et al. Clinical Immunology, 2002, 104:105-114. The peptide was placed into limited human trials, which reportedly resulted in the long term immune reactivity against the peptide, but the treatment has been deemed clinically ineffective by evaluation using MRI. Thus this APL, as with many antibody-based therapeutic candidates, had limited effectiveness in terms of clinical efficacy.
Further complicating the application of the technology, an APL that may induce an antagonist-like reaction, may also induce a partial agonist response, or induce a state of anergy in the reactive T cell population. See, for example, Fairchild et al., Curr. Topics Peptide Protein Res. 2004, 6:237-44, who, in discussing APL in the context of allograft rejection therapy, note that an APL acting as an antagonist for one TCR, may become an agonist for another.
The approach using APL, along with other approaches currently known in the art, to identify therapeutic peptides, while recognizing the advantage of variations in the therapeutic peptide compositions, derive from the concept that there is one or more defined peptide sequence evoking a defined immunological response. These strategies have attempted to multiply and diversify modulatory peptides via the introduction of defined, single changes performed one at a time.
An entirely different approach which has evolved alongside the defined sequence peptide immunotherapy approach is the use of limited amino acid diversity, random epitope polymers. Random sequence polymers (RSP) can be described as a random order mixture of amino acid copolymers comprising two or more amino acid residues in various ratios, forming copolymers by random sequence bonding, preferably through peptide bonds, of these amino acid residues, which mixture is useful for invoking or attenuating certain immunological reactions when administered to a mammal. Because of the extensive diversity of the sequence mixture, a large number of therapeutically effective peptide sequences are likely included in the mixture. In addition, because of the additional peptides which may at any given time not be therapeutically effective, but may emerge as effective as the epitope shifting and spreading occurs, the therapeutic composition may remain effective over a time of dosing regimen. This approach is schematically represented in FIG. 1D.
Copolymer-1 (also known as Copaxone, glatiramer acetate, COP-1, or YEAK random copolymer), is used for the treatment of multiple sclerosis. Random copolymers are described in International PCT Publication Nos. WO 00/05250, WO 00/05249; WO 02/59143, WO 0027417, WO 96/32119, WO/2005/085323, in U.S. Patent Publication Nos. 2004/003888, 2002/005546, 2003/0004099, 2003/0064915 and 2002/0037848, in U.S. Pat. Nos. 6,514,938, 5,800,808 and 5,858,964. Copolymer-1 has been used in combination with a mucosal adjuvant and an A beta peptide for the development of an Alzheimer's vaccine (Frenkel, Dan et al., 2005, J Clin Invest., 115:2423), and has been described as a constituent in a method of vaccination designed to regenerate neuronal tissue (U.S. Pat. No. 6,844,314).
Tracing back steps to the defined peptide search, there have also been attempts to identify the active peptide(s) within the RSP mixture. The drawback of this technology lies in the very nature of the attempt to determine discrete substitutes for the randomness that COP-1 encompasses.
Effective as the random sequence polymer approach may be, even the improvements have not resolved the drawback and limitation of COP-1, which is, for example, the undefined nature of what is effective in each motif and the possibility of containing a large proportion of truly inactive peptides, lowering the concentration of the active components, or worse, adversely stimulating the immune system. Additionally, these compounds are difficult to manufacture and to obtain consistency from lot-to-lot.
Despite the modest success of the existing approaches, need remains for a composition and a method to create such composition that would serve effectively as a vaccine and immunogen by eliciting beneficial immune responses consistently and over time toward pathological proteins or peptides related to a PCD, for which existing vaccine compositions have failed to be effective.